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Biomedical subjects

W Salser

Publications and source records attributed to W Salser.

At least 19 recordsLinked to original sources
PubMed1979

Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain.

The recombinant plasmid pH21-1 consists of mouse-derived complementary DNA (cDNA) in the E. coli plasmid pMB9. The mouse insertion has been completely sequenced, and encodes the CH3 domain and half the CH2 domain of the immunoglobulin gamma1 heavy chain. The predicted amino acid sequence differs at several positions from that previously published for this protein. The pattern of codon usage resembles that in some other eukaryotic messenger RNAs. A computer program has been used to predict the optimum secondary structure for the mRNA encoding the CH3 domain and the inter-domain junction.

Animals↗
PubMed1979

Isolation of human germ-line DNA suitable for recombinant DNA studies.

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.

Centrifugation, Density Gradient↗
PubMed1978

Characterization of an immunoglobin cDNA clone containing the variable and constant regions for the MOPC 21 kappa light chain.

Nucleotide sequence analysis and restriction endonuclease mapping have been used to characterize a cDNA copy of immunoglobulin MOPC 21 Kappa mRNA clones in the bacterial plasmid pMB9. Three regions of the inserted cDNA of plasmid pL21-1 have been sequenced and match the known protein sequence at amino acid residues 1-24, 128-138 and 171-179. With these sequences to provide absolute correlations between the restriction map and the structural gene sequence it has been possible to exactly deduce the positions of all 11 of the insert restriction sites mapped within the structural gene. The pL21-1 insert contains the complete variable and constant regions as well as parts of the 3' untranslated and polypeptide leader coding sequences.

Amino Acid Sequence↗
PubMed1978

Recombinant DNA clones constructed from immunoglobulin kappa light chain messenger RNA.

Recombinant DNA clones have been generated from mouse myeloma MOPC 21 immunoglobulin kappa light chain mRNA. Complementary DNA (cDNA) synthesized on kappa light chain mRNA by reverse transcriptase was made double stranded and inserted into the bacterial plasmid vector, pMB9. Approximately 70 tetracycline-resistant transformed colonies containing kappa light chain mRNA sequences were identified by colony hybridization. Five of these recombinant clones were selected and characterized. Three clones contain both kappa light chain constant and variable region sequences. Two of these three recombinant clones have been shown to include all of the kappa light chain constant and variable region coding sequences. Another of the five selected recombinant clones contain kappa light chain constant region sequences. The remaining characterized clone appears to be derived from sequences at the 5'-end of kappa light chain mRNA, possibly extending to the terminal cap structure.

Base Sequence↗
PubMed1977

Rabbit globin mRNA: analysis of T1 RNAse digestion fragments.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.

Amino Acid Sequence↗
PubMed1977

Nucleotide sequences from a rabbit alpha globin gene inserted in a chimeric plasmid.

Rabbit alpha globin gene copies have been made, using reverse transcriptase and DNA polymerase I, and cloned in bacterial plasmids. Plasmid pHb72 has been shown to contain the alpha gene sequence by restriction enzyme analysis and nucleotide sequencing studies, and therefore has been approved for propagation under P2 plus EK1 conditions by the National REcombinant DNA Committee.

Base Sequence↗
PubMed1977

The ovalbumin gene. Insertion of ovalbumin gene sequences in chimeric bacterial plasmids.

Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants. The double-stranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript. After S. nuclease treatment, poly(dA) was added to the 3' termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3'-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation of the fragments by agarose gel electrophoresis showed that the amount of inserted DNA in clones tested varied from 680 to 1090 base pairs.

Animals↗
PubMed1976

Optical measurements reveal base-pairing in T4-specific mRNAs.

Previous studies have shown that 60-70% of the nucleotides in rRNA, tRNA and the RNA of the RNA bacteriophages are involved in base pairing. To determine whether this is also true of mRNAs coded from double-stranded DNA, T4-specific mRNAs were purified using two different techniques: hybridization to T4 DNA or chromatography on benzoylated DEAE-cellulose columns followed by sucrose gradients. We have determined the optical melting curves of the purified mRNAs. The hypochromicities calculated from these melting curves were similar to those of rRNA, indicating comparable amounts of base pairing in rRNA and T4-specific mRNA. If this hypochromicity were due to random base pairing, we would have expected a less sharp melting curve and a lower Tm than for the rRNA controls. On the contrary, the mRNA samples had very high cooperativities and Tm values.

Base Sequence↗
PubMed1976

A general method for cloning eukaryotic structural gene sequences.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Base Sequence↗
PubMed1976

Investigation of the organization of mammalian chromosomes at the DNA sequence level.

New developments in DNA sequencing techniques permit rapid progress in the determination of both repetitious and single-copy mammalian sequences. Three distinct families of highly repetitious satellite DNA's from the kangaroo rat Dipodomys ordii have been sequenced. With the MS satellite it was possible to show that the basic repeat sequence and its variants were arranged in a nonrandom order suggesting a hierarchy of repeats. The HS-alpha satellite from D. ordii was shown to resemble the guinea pig alpha satellite, a long term evolutionary persistence inconsistent with previous models. Sequences from hemoglobin mRNA were determined using hemoglobin complementary DNA as template for transcription in vitro. Seven of the largest fragments have been assigned to untranslated regions of the mRNA whereas 15 others have been tentatively located within the structural genes. From correlations with sequences from corresponding regions in the human hemoglobin mRNA's we have been able to make the first direct measurements of the rate of fixation of mutations that do not change the amino acid sequence. The minimum estimate for this rate is greater than the highest previously estimated rates of fixation of neutral mutations (calculated for fibrinopeptide A. A new technique, deoxysubstitution sequencing, which should speed determination of the complete mRNA sequences, is described.

Animals↗